To assess how rap-idly SE enhances the infectiousness of HIV-1, we mixed virus stocks with various concentrations of SE and used these cocktails to infect TZM-bl indicator cells after di
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R E S E A R C H
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Research
Semen-mediated enhancement of HIV infection is donor-dependent and correlates with the levels of SEVI
Kyeong-Ae Kim†1, Maral Yolamanova†1, Onofrio Zirafi1, Nadia R Roan2, Ludger Staendker3, Wolf-Georg Forssmann3,4, Adam Burgener5, Nathalie Dejucq-Rainsford6, Beatrice H Hahn7, George M Shaw7, Warner C Greene2,
Frank Kirchhoff*1 and Jan Münch*1
Abstract
Background: HIV-1 is usually transmitted in the presence of semen We have shown that semen boosts HIV-1 infection
and contains fragments of prostatic acid phosphatase (PAP) forming amyloid aggregates termed SEVI (semen-derived enhancer of viral infection) that promote virion attachment to target cells Despite its importance for the global spread
of HIV-1, however, the effect of semen on virus infection is controversial
Results: Here, we established methods allowing the meaningful analysis of semen by minimizing its cytotoxic effects
and partly recapitulating the conditions encountered during sexual HIV-1 transmission We show that semen rapidly and effectively enhances the infectivity of HIV-1, HIV-2, and SIV This enhancement occurs independently of the viral genotype and coreceptor tropism as well as the virus producer and target cell type Semen-mediated enhancement of HIV-1 infection was also observed under acidic pH conditions and in the presence of vaginal fluid We further show that the potency of semen in boosting HIV-1 infection is donor dependent and correlates with the levels of SEVI
Conclusions: Our results show that semen strongly enhances the infectivity of HIV-1 and other primate lentiviruses
and that SEVI contributes to this effect Thus, SEVI may play an important role in the sexual transmission of HIV-1 and addition of SEVI inhibitors to microbicides may improve their efficacy
Background
Since its introduction into the human population in the
first half of the 20th century by zoonotic transmission of
simian immunodeficiency viruses (SIVs) found in
chim-panzees [1], HIV-1 has caused one of the most
devastat-ing pandemics of modern times To date, HIV-1 has
infected more than 60 million people and caused about
25 million deaths [2] In 2008 alone, there were 2.7
mil-lion new HIV-1 infections and 2.0 milmil-lion AIDS-related
fatalities The great majority of all HIV-1 transmissions
results from unprotected sexual intercourse Despite the
rapid spread of HIV-1, the estimated rate of transmission
per sexual intercourse is surprisingly low: male to female
virus transmission occurs about once in every
1,000-10,000 unprotected vaginal act [3,4] Receptive anal
inter-course carries an approximately 10-fold higher risk [5] Furthermore, the rate of transmission is affected by the viral load in the donor and thus high during acute HIV-1 infection [6] Furthermore, inflammation and lesions in the mucosal barrier resulting from other sexually trans-mitted diseases increase the risk of transmission [7] Nonetheless, the dose of HIV-1 transmitted during sexual intercourse is usually subinfectious and clearly limiting viral spread [8]
Genital exposure to semen (SE) contaminated with HIV-1 accounts for most transmissions worldwide [9] Thus, SE represents the major vector for the dissemina-tion of HIV-1 Several intrinsic properties of SE might affect the efficiency of HIV-1 transmission, such as neu-tralization of the acidic pH of the vaginal fluid [10], stim-ulation of inflammatory cytokines [11], and induction of leukocyte infiltration of the cervical mucosa and migra-tion of Langerhans cells [12,13] All these effects may
pro-* Correspondence: frank.kirchhoff@uni-ulm.de, jan.muench@uni-ulm.de
Institute of Molecular Virology, University Hospital Ulm, 89081 Ulm, Germany
† Contributed equally
Full list of author information is available at the end of the article
Trang 2mote HIV-1 transmission by indirect mechanisms It is
less clear, however, whether SE directly affects the
infec-tiousness of HIV in male genital fluid For example, it has
been reported that seminal plasma (SE-P) contains
fac-tors preventing the capture and transmission of HIV-1 to
CD4+ T cells by DC-SIGN expressed on dendritic cells
[14] Another study reported that SE-P contains cationic
polypeptides that inhibit HIV-1 infection [15] On the
other hand, spermatozoa themselves may capture HIV-1
through heparan sulfate and efficiently transmit the virus
to dendritic cells [16]
We have previously shown that fragments of the
abun-dant semen protein prostatic acidic phosphatase (PAP)
form amyloid structures that capture HIV-1 virions and
promote their attachment to target cells [17] Strikingly,
these amyloid aggregates, termed Semen-derived
Enhancer of Virus Infection (SEVI), enhance the
infec-tious titer of HIV-1 by several orders of magnitude at a
low multiplicity of infection [17] The structure of
PAP248-286 (numbers refering to the amino acid
posi-tion in PAP), the predominant enhancing PAP fragment
detected in semen, has recently been solved and its has
been confirmed that this peptide is highly amyloidogenic
[18,19] The mechanism underlying SEVI-mediated
enhancement of HIV-1 infection most likely involves
nucleation-dependent formation of amyloid aggregates
and a direct interaction of positively charged surfaces of
SEVI with negatively charged membranes [17,20,21]
SEVI seems to promote virion attachment and
subse-quent infection by allowing the virus to overcome the
electrostatic repulsion between the negatively charged
viral and cellular membranes Notably, SEVI and SE also
boost the infectiousness of XMRV (xenotropic murine
leukemia virus-related virus), a novel γ-retrovirus that
may be associated with prostate cancer and chronic
fatigue syndrome [22] and SEVI increases the efficiency
of retroviral transductions [23]
The ability of SE and SEVI to promote HIV-1 infection
has been confirmed in several studies [17,20,22,24,25]
Furthermore, the first inhibitors of SE- and
SEVI-medi-ated enhancement of HIV-1 infection have been reported
[24,25] and may lead to new approaches to prevent HIV-1
transmission Despite its possible importance in the
transmissions of HIV/AIDS, however, the enhancing
effect of SE on HIV-1 infection remains poorly defined
and controversial One reason is the lack of standardized
methods addressing the high cytotoxicity of SE in in vitro
culture systems Here, we thus established methodologies
allowing the meaningful analysis of SE by minimizing its
cytotoxic effects The results show that SE rapidly and
effectively enhances HIV-1 infection independently of the
viral strain and/or the virus producer or target cell type
Altogether, our data further support an important role of
SEVI in SE-mediated enhancement of HIV-1 infection
and thus in the transmission of HIV/AIDS
Results
Semen boosts HIV-1 infection under non-cytotoxic conditions
It is long known that the intrinsic cytotoxicity of SE com-plicates its meaningful analysis in cell culture [17,26] Thus, we first established experimental conditions cir-cumventing this problem Specifically, we reduced the final concentrations of SE in cell culture by pre-incubat-ing SE with the HIV-1 virions (rather than the target cells) and adding small volumes (usually 20 μl) of these HIV/SE mixtures and serial dilutions thereof, to comparatively large (typically 280 μl) TZM-bl cell cultures (Figure 1A)
In some aspects this approach resembles sexual transmis-sion of HIV, where virus-containing SE is diluted by the female genital fluid present in the vaginal tract, which has
a relatively large surface area of about 100 cm2 [27] To further reduce cytotoxicity we removed the SE containing medium after 2 hours and cultured the cells in fresh medium containing gentamicin (to prevent bacterial out-growth) until HIV-1 infection was assessed 2 to 3 days later Under these conditions pre-treatment of virions with 90% (v/v) SE enhanced HIV-1 infection up to 40-fold compared to the untreated control (Figure 1B) In con-trast, PBS-treated HIV-1 was more infectious than SE-treated virus when the inoculum was left on the target cells (Figure 1B) because final concentrations of SE as low
as 1% were cytotoxic (Figure 1C) Thus, the cytotoxicity
of SE may mask enhancing effects and produce mislead-ing results, but can be overcome by reducmislead-ing the concen-tration of SE and exposure time
Further experiments showed that even SE concentra-tions as low as 0.4% during virion treatment markedly enhance HIV-1 infection (Figure 1D) Exposure to rela-tively high doses of SE-treated HIV-1 caused massive cytopathic effects (examples shown in Additional file 1 Figure S1) Under these conditions the detectable levels
of LTR-driven reporter gene activity saturated or even declined due to over-infection and virus-mediated cell killing Thus, HIV-1 infection rates and the effects of SE can only be faithfully determined at a relatively low multi-plicity of infection Notably, SE also enhanced infection when the HIV/SE inoculum was not removed if the size
of the target cell cultures was increased to further mini-mize cytotoxic effects (Additional file 1 Figure S2) The ability of SE to promote HIV-1 infection was not affected
by gentamicin and did not require a serum cofactor (Additional file 1 Figure S3) Furthermore, treatment with
SE alone did not induce LTR-dependent reporter gene expression (data not shown)
SE enhances HIV-1 infection rapidly and at different pH conditions
SE is composed of secretions from different sources, including the epididymis (~5% v/v of the fluid), seminal vesicles (60%), prostate (20-30%), and bulbourethral
Trang 3glands The origin of seminal HIV-1 particles is still
unclear [28] but they are at least in part produced within
the male genital tract [29-31] Thus, virions may be
exposed to enhancing factor(s) in SE, such as fragments
of PAP produced by the prostate gland, an organ
produc-tively infected by HIV/SIV [30,31], just immediately prior
to their deposition in the genital tract To assess how
rap-idly SE enhances the infectiousness of HIV-1, we mixed
virus stocks with various concentrations of SE and used
these cocktails to infect TZM-bl indicator cells after
dif-ferent incubation periods We found that SE enhances
HIV-1 infection, even when the HIV/SE solution was
added to the cells immediately after mixing (Figure 2A)
The highest levels of HIV-1 infection were usually
mea-sured after treatment with 10% SE because 50% SE
(corre-sponding to 3.3% in the cell culture) frequently caused
cytotoxic effects (Figure 2A and data not shown) To
assess whether the effect of SE on HIV-1 infection
depends on the duration of target cell exposure, we
infected TZM-bl cells with untreated or SE-treated
HIV-1 and removed the inoculum after different incubation
periods We found that the efficiency of HIV-1 infection
was low during the earliest time points and increased
with longer exposure times (Figure 2B) This was
expected because virus entry may take several hours, and
unbound or loosely attached HIV-1 virions are removed
during the washing step Importantly, SE promoted
HIV-1 infection at all time points analyzed However, the effect
was most pronounced (up to 40-fold) between 1 and 4
hours of virus exposure (Figure 2B) Shorter time periods
resulted in inefficient viral infection and longer
incuba-tion increased cytotoxic effects
Next, we examined the effect of different pH conditions
on SE-mediated enhancement of HIV-1 infection This may be relevant for sexual transmission of HIV-1 because the pH in the viral environment prior to, during and after sexual intercourse may change from about 8.0 in SE to about 4.2 in the female genital fluid [10] Unexpectedly, HIV-1 infection was moderately increased at an acidic pH
in target cell cultures (Figure 2C), whereas the pH of the virus stock had no significant effect on the efficiency of HIV-1 infection (Figure 2D) Importantly, SE boosted virus infection under pH conditions ranging from 5.5 to 8.0 and in the presence of pooled cervical lavage fluid (CLF) (Figure 2E) More extreme pH conditions or increased CLF concentrations could not be analyzed, as they were toxic to the cells
Stability of the enhancing activity in SE
To assess the stability of the enhancing activity in SE, we incubated aliquots of pooled SE for three days at 37°C and tested its effect on HIV infection We found that incuba-tion of SE for 6 hours at 37°C reduced its enhancing activ-ity by about 50% (Additional file 1 Figure S4A) Some residual activity was even detectable at 3 days of incuba-tion (Addiincuba-tional file 1 Figure S4A) After sexual inter-course, elevated levels of the SE marker PAP, the precursor of SEVI, can be detected in the vagina for about
24 hours [32] Thus, amyloidogenic PAP fragments may
be generated over a period of several days Notably, 10%
SE was most effective in infectivity enhancement for the first 6 hours, whereas 50% SE showed higher activity at later time points This indicates that the enhancing and cytotoxic factors in SE are slowly degraded Furthermore, both activities were eliminated by heating (data not
Figure 1 Effect of SE on HIV infection (A) Schema describing the experimental procedure (B) Effect of treatment of virus stocks with 90% (v/v) of
SE on R5 HIV-1 infection TZM-bl cells were infected with the indicated dilutions of SE- or PBS-treated virus stocks The inoculum was either removed after 2 hours of exposure (wash) or left on the cells Shown are average β-galactosidase activities (n = 3) measured 2 days after virus exposure RLU/s: relative light units per second The numbers above the upper curve give n-fold enhancement of HIV infection by SE relative to that measured for the corresponding PBS control (C) Metabolic activities of cells analysed in B (D) Effect of low concentrations of SE on HIV infection R5 HIV-1 stocks were treated with the indicated concentrations of SE, diluted and used to infect TZM-bl cells The Y-axis gives average values of triplicate infections, and the X-axis gives the final dilution of the virus stocks The infection levels were determined as described above Percentages refer to the SE concentrations during virion treatment The final concentrations in the cell culture are 15-fold lower.
Trang 4shown), suggesting that they may represent peptides or
proteins Most importantly, these data show that SE
maintains some enhancing activity for several days at
body temperature
SE generally enhances primate lentiviral infection
All of the results described above were obtained using
adherent TZM-bl cells allowing easy removal of the SE
inoculum To assess the effect of SE on HIV-1 infection in
T cells, we exposed CEMx-M7 cells to SE- and
SE-F-treated CXCR4(X4)- and CCR5(R5)-tropic HIV-1 strains
Examination by fluorescence microscopy and flow
cytometry confirmed that treatment of HIV-1 virions
with 10% SE and SE-F increased the number of GFP+
infected cells up to 17-fold (Figure 3A and Additional file
1 Figure S5A and S5B) Prior to their deposition in the
genital tract, HIV-1 virions are exposed to undiluted SE
Thus, it is noteworthy that the infectiousness of HIV-1
particles was also enhanced up to 20-fold by treatment with 90% SE (Additional file 1 Figure S5C and S5D) Unexpectedly, CEMx-M7 cells infected with SE- or SE-F-treated virions also displayed about 2- to 3-fold increased levels of mean GFP expression compared to those infected with PBS-treated virus (Additional file 1 Figure S5B and S5D)
Next, we investigated whether the enhancing effect of
SE on HIV-1 infection is also observed in primary human cells To address this, we generated virus stocks of wild-type X4 HIV-1 NL4-3 and twenty V3 Envelope recombi-nants thereof with differential coreceptor tropism [33] These viruses were exposed to PBS or to 10% (v/v) SE for
5 min prior to infection A total of 20 μl of these virus stocks was then used to infect 280 μl PBMC cultures After 3 days, the cell-free PBMC culture supernatants were used to infect TZM-bl cells (experimental outline shown in Figure 3B) To determine the effect of SE on PBMC infection and release of HIV-1 we analyzed virus production at an early time point to avoid a bias due to multiple rounds of viral replication Strikingly, treatment with SE resulted in 5- to 137-fold (average 36.1 ± 32.7-fold, n = 21) enhancement of infectious virus in the supernatant of the PBMC cultures (Figure 3B) Effective enhancement was observed for the 16 R5 viruses as well
as for the three X4 and two dual tropic HIV-1 recombi-nants, suggesting that SE-mediated enhancement is
inde-Figure 3 The enhancing effect of SE is independent of the viral coreceptor tropism (A) Analysis of CEMx-M7 cells infected with
un-treated or SE-un-treated (10% v/v) X4 and R5 HIV-1 by fluorescence mi-croscopy 2 dpi (B) Treatment with SE enhances HIV-1 infection of primary PBMCs Stimulated PBMCs were infected with the same dose
of the indicated HIV-1 NL4-3 V3 recombinants that were either not treated or treated with 10% (v/v) SE Three days later, 100 μl of the cell-free PBMC culture supernatant was used to infect TZM-bl indicator cells Shown are average infection rates of TZM-bl cell ± SD (n = 3) mea-sured 2 days after virus exposure X4 virus is color-coded red; R5 virus, green; and X4R5 HIV-1, black.
Figure 2 Effect of exposure times and pH values on SE mediated
enhancement of HIV infection (A) Effect of pre-incubation time on
SE-mediated HIV infection enhancement R5 HIV-1 was mixed with the
indicated concentrations of SE, incubated for the various time periods,
and 20 μl of the virus stocks was used to infect 280 μl TZM-bl cell
cul-tures in triplicates Values in all panels give averages ± SD (n = 3)
mea-sured 3 days after virus exposure (B) The SE/virus mixture was
incubated for 10 min at RT, and 20 μl were added to 280 μl TZM-bl cells
After different time points, the supernatant was removed, and fresh
DMEM was added for further culture The star indicates high
cytotoxic-ity (C) Virus stocks of R5 HIV-1 treated with the indicated
concentra-tions of SE were used to infect TZM-bl cultures adjusted to the
indicated pH values After two hours of virus exposure, the virus stocks
were removed, and the cells were cultured in fresh medium under
neutral pH conditions Higher or lower pH values could not be
ana-lyzed as they were cytotoxic Both panels give average values ± SD (n
= 3) (D) Virus stocks adjusted to the indicated pH values were either
treated with PBS or with various concentrations of SE and
subsequent-ly used to infect TZM-bl indicator cells (E) TZM-bl cells were incubated
with either PBS or 10% cervico vaginal lavage (CLF) and infected with
medium or 10% SE treated HIV-1 Infection rates were determined 3
dpi.
Trang 5pendent of the viral coreceptor tropism To further
examine whether SE might generally enhance primate
lentiviral infection, we analyzed its effect on a large
num-ber of HIV-1, HIV-2 and SIV strains and found that SE
enhanced their infectiousness by 10- to 25-fold
(Addi-tional file 1 Figure S6 and data not shown)
SE-mediated enhancement of HIV-1 infection is
independent of the virus producer and target cell type
To assess the possible relevance of SE for sexual virus
transmission, we next examined its effect on HIV-1
founder viruses, which are most likely the ones
transmit-ted during sexual intercourse [34] We found that SEVI
and pooled SE enhance the infectiousness of HIV-1
parti-cles pseudotyped with envelope glycoproteins derived
from 25 different transmitted/founder viruses [34] in
sin-gle round infection assays by 5- to 48 fold (Figure 4A)
Notably, the magnitudes of infectivity enhancement by
SEVI and SE correlated significantly (p = 0.0006) (Figure
4B) To further examine the effect of SE on viral particles
generated by the relevant producer cells in vivo we
har-vested HIV-1 from ex vivo infected testis tissue [29,31].
We found that SE clearly increases the infectiousness of
testis-derived R5- and X4-tropic HIV-1 strains (Figure
4C) Further experiments using luciferase reporter
viruses showed that SE also promotes HIV-1 infection of
primary T cells and macrophages, the relevant target cells
of HIV-1 in vivo (Figure 4D and 4E) Finally, we examined
whether SE also affects HIV-1 infection in trans Our
results showed that SE increases the transmission of
R5-tropic HIV-1 from non-permissive CaSki human cervical
epithelial carcinoma cells to susceptible T cells about
80-fold (Figure 4F) Typically, the strongest enhancing effects
of SE were observed with low doses of freshly produced
highly infectious HIV-1 virions, irrespective of the virus
strain and producer or target cell type
Exploring controversies on the effect of SE on HIV-1
infection
Our result that SE enhances HIV-1 infection seems to be
contradictory to previous studies reporting that seminal
plasma (SE-P) impairs the capture and transmission of
HIV-1 by DC-SIGN [14] and inhibits virus infection [15]
To determine the effect of SE and SE-P on HIV-1
trans-mission by DC-SIGN we used B-THP-1-DC-SIGN and
CEM-M7 cells As expected [14], expression of DC-SIGN
strongly enhanced transmission of HIV-1 to CEM-M7
indicator cells (Figure 5) In agreement with previous
results [14], pre-treatment of cells with SE, SE-F and SE-P
potently inhibited DC-SIGN-mediated transmission of
HIV-1 (Figure 5) In contrast, SEVI amplified infection of
T cells by HIV-1 particles bound to DC-SIGN-expressing
dendritic or B-THP-1 cells even further [[17], data not
shown] Thus, SEVI generally facilitates HIV-1 infection, whereas SE also contains a specific inhibitor that over-comes the enhancing effect of SEVI in the case of DC-SIGN-mediated virus transmission
Next, we evaluated results of Martellini and coworkers suggesting that SE-P may inhibit HIV-1 [15] In this study, the target cells and not the HIV-1 virions were treated with SE-P To assess the effect of SE on the susceptibility
of target cells to HIV-1 infection, we used a flow
cytome-Figure 4 SE enhances founder HIV infection and boosts HIV infec-tion independently of the viral producer and target cell type (A)
Effect of SE on HIV particles carrying gp120/41 from founder viruses Pseudotyped HIV-1 particles were generated by transient transfection
of 293T cells with an env-defective HIV-1 NL4-3 backbone and plasmids
expressing the Env proteins previously described (34) Virions were treated with medium, 10 μg/ml SEVI or 10% SE and used to infect
TZM-bl cells The inoculum was removed after 2 hrs and infection rates were determined 2 dpi Shown are the average levels of triplicate TZM-bl cell infections ± SD (B) Correlation between the magnitudes of SEVI and SE-mediated enhancement of HIV-1 pseudotype infection shown in Fig 4A N-fold enhanced infection rates were calculated by dividing in-fection rates obtained in the presence of SEVI or SE by those of mock-treated virus infection (C) SE enhances infection of testis derived
HIV-1 X4 tropic HIV-1 IIIb and R5 tropic SF162 were harvested from infected testis tissue, treated with indicated concentrations of SE and used to infect TZM-bl cells (D, E) SE enhances the infectiousness of HIV-1 for PBMCs and macrophages Stocks of an R5-tropic HIV-1 NL4-3 V3 variant
(92TH04.12) containing the luciferase reporter gene in place of nef
were generated by transient transfection of 293T cells Virus stocks were treated with the indicated concentrations of SE and used to in-fect PBMC (D) and macrophages (E) Similar results were obtained
us-ing various primary HIV-1 strains (F) SE favours in trans-infection of T
cells by viral particles bound to epithelial cells CaSki cells derived from
an epithelial cervical carcinoma were exposed to HIV-1 treated with SE
or medium for 3 hrs Subsequently, the virus inoculum was washed out and the cells were cocultivated with CEM-M7 cells for three days Infec-tion rates were determined by luciferase assay The numbers above the bars indicate n-fold enhancement relative to the infectivity measured using PBS/medium-treated virus stocks.
Trang 6try-based HIV-1 virion-fusion assay [35] This system
allows to directly measure virion fusion with the target
cells and minimizes cytotoxic effects because the cells are
only exposed to SE for short time periods Primary
endo-metrial CD4+ T cells were either PBS-treated or treated
with SE, washed, exposed to HIV-1 NL4-3 BlaM-Vpr
viri-ons for 4 hours and loaded with CCF2/AM dye In
agree-ment with published data [20,24], treatagree-ment with 10% SE
enhanced the susceptibility of the cells to HIV-1 infection
by 5-fold (Additional file 1 Figure S7) This effect is lower
than that observed in HIV-1 infection assays because the
fusion assay requires higher viral doses Nonetheless, our
data show that treatment with SE enhances rather than
reduces the susceptibility of cells to HIV-1 infection To
elucidate why Martellini and coworkers obtained
differ-ent results, we repeated the experimdiffer-ents following their
protocols (Figure 6A) In agreement with their findings,
treatment of the TZM-bl indicator cells with SE-P and SE
and subsequent infection with HIV-1 resulted in reduced
levels of Tat-driven reporter gene activity suggesting
inhi-bition of virus infection (Figure 6B) At one day
post-infection (the time point examined in the previous study)
cytotoxic effects were observed at about 4-fold higher
concentrations of SE-P and SE compared to those
required to inhibit virus infection (Figure 6C, upper) At 3
days post-infection cytotoxic effects become more
appar-ent (Figure 6C, lower) and the metabolic activity of the
cells correlated significantly with the Tat-driven reporter
gene activities (Figure 6D) Thus, a decreased metabolic
activity of the target cells rather than a specific anti-HIV
activity of SE-P or SE may account for the reduced levels
of Tat-driven reporter gene activity in this assay Efficient
viral gene expression is dependent on the "fitness" of the
cells and is highly sensitive to cytotoxic or cytostatic
effects and experimental conditions just at the threshold
of cytotoxicity may yield misleading results
SE-mediated enhancement of HIV-1 infection is donor-dependent and correlates with the levels of PAP248-286/ SEVI
All experiments described above were performed with pooled semen obtained from more than 20 individual donors To assess whether the enhancing activity may be donor-dependent, we collected and analyzed SE samples from 14 different individuals All SE samples were allowed to liquefy for 30 min and subsequently kept fro-zen until further use Notably, they were processed and tested together for their ability to promote HIV-1 tion We found that all SE samples enhanced HIV-1 infec-tion, albeit with strikingly different efficiencies ranging from 2- to about 50-fold (Figure 7A) Thus, in addition to the viral load and other factors, the potency of SE in enhancing the infectiousness of HIV-1 particles may also affect the rates of virus transmission Notably, fresh lique-fied ejaculates enhanced HIV-1 infection about as effec-tively as SE samples that had undergone a freeze/thaw cycle (data not shown) Thus, the presence of living sper-matozoa capturing virions by heparan sulfate [16] or other cells did not interfere with SE-mediated enhance-ment of HIV infection
To assess a possible role of SEVI in the differential capacity of these SE samples to enhance HIV-1 infection,
we generated specific antibodies by immunizing rabbits and guinea pigs with amyloidogenic PAP248-286 Both immune sera (but not the pre-immune sera) reacted with monomeric PAP248-286 and with SEVI amyloid, but not with shorter PAP fragments (e.g PAP248-261), a peptide containing the reverse amino acid sequence of
PAP248-286 (termed "IVES"), or with full-length PAP (Figure 7B
Figure 6 Adding SE or SE-P directly to target cells results in re-duced metabolic activity and HIV infection rates (A) Schematic
outline of the experiment TZM-bl cells were incubated with different dilutions of SE or SE-P and subsequently infected with HIV-1 (B) Infec-tion rates and (C) metabolic activities were determined after 1 (upper panel) or 3 days (lower panel) (D) Correlation between Tat-driven re-porter activities ("infection") and the metabolic activities of the target cells Values were derived from the experiments shown in panels B and
C, and are shown relative to those obtained in the absence of SE or
SE-P (100%).
Figure 5 Semen inhibits trans-infection of T cells by DC-SIGN
B-THP-1-DC-SIGN cells were treated with the indicated concentration of
SE, SE-F or SE-P for 30 min, subsequently exposed to R5 HIV-1 for 30
min, washed and cocultivated with CEM-M7 cells The levels of
infec-tion mediated by B-THP-1 cells, which do not express DC-SIGN, and the
absence of cells (medium) are also shown as controls Shown are
aver-age values ± SD derived from triplicate infections Stars indicate
cyto-toxicity, infection rates obtained after infection with 2% and 10% SE,
SE-F or SE-P treated virus were close to background luciferase activities
of uninfected cells.
Trang 7and 7C, and data not shown) Importantly, the sera also
recognized SEVI spiked into SE (Figure 7D) and allowed
to establish a semi-quantitative indirect "SEVI ELISA"
The potency of the 14 SE samples described above in
enhancing HIV-1 infection correlated significantly with
their reactivity to the anti-SEVI sera (Figure 7E) This
finding was confirmed with a large number of SE samples
from both Germany and the US (data not shown)
Nota-bly, centrifugation of SE-F through a 100-kDa-pore-size
filter removed the entire virus enhancing activity and the
reactivity to anti-SEVI antiserum (Additional file 1
Fig-ures S4B and S4C) Thus, the enhancing factor in SE has a
molecular weight of > 100 kDa, which is in agreement
with the large size of amyloid aggregates Altogether,
these data are further evidence that SEVI contributes to
SE-mediated enhancement of HIV-1 infection
Further-more, they emphasize the importance of using pooled SE
samples for a valid analysis of SE-mediated enhancement
of HIV-1 infection because the activity of individual
sam-ples varies substantially
Discussion
SE is the main vector for the spread of the AIDS
pan-demic [9] and contains cell-free HIV-1 virions, even in
individuals receiving HAART [36,37] It is well
estab-lished that the levels of infectious virus transmitted dur-ing sexual intercourse are a major determinant of the rate
of HIV-1 transmission [6,38] Nonetheless, strikingly lit-tle is known about the effect of SE on the infectiousness
of HIV-1 Our results suggest that the ability of SE to boost HIV-1 infection may have been missed because its meaningful analysis is not without complications We show that the confounding effects of cytotoxic factors in
SE can largely be overcome by (i) pre-treating the virus rather than the cells; (ii) using a small volume of SE-treated virus stocks to infect a large target cell culture and (iii) removing the HIV/SE inoculum after a few hours While the analysis of SE for enhancement of HIV-1 infec-tion requires some optimizainfec-tion, these condiinfec-tions actu-ally partly recapitulate those encountered during sexual transmission of HIV-1 Other treatments commonly used
to examine SE, such as pre-incubation of target cell, heat treatment and infection with high concentrations of freeze-thawed viral inoculums, all reduce the ability of SE
to enhance HIV-1 infection, or may even misleadingly suggest that it is inhibitory
The newly established experimental conditions allowed
to demonstrate that SE promotes HIV-1 infection inde-pendently of the viral geno- and phenotype or the viral producer and target cell type Importantly, our analyses included HIV-1 founder viruses as well as the primary
cell types relevant for virus production and infection in
vivo (Figure 4A) The enhancing effect of SE was relatively stable (Additional file 1 Figure S4) and observed over a broad range of pH conditions as well as in the presence of vaginal fluid (Figure 2C-E) Our finding that SE increases the infectiousness of HIV-1 almost instantaneously (Fig-ure 2A) suggests that it may also affect the rate of female-to-male virus transmission, particularly if mixtures of HIV-1 containing vaginal fluid and semen become stuck under the foreskin Notably, the observed effects most likely underestimate the potency of SE in boosting HIV-1 infection because its cytotoxicity precluded the analysis
of high SE concentrations or long cellular exposure times Our previous screening of a highly complex protein/ peptide library from pooled SE identified only enhancing amyloidogenic peptides, but no inhibitors of HIV [17] However, we also confirmed findings of Sabatte and coworkers [14] showing that pretreatment of DC-SIGN expressing cells with SE inhibits the transmission of
HIV-1 to CD4+ target cells This inhibitor could not be detected in our screening approach because its molecular mass exceeds the cut-off size used for the generation of the SE-derived peptide/protein library and because we examined effects on CD4/coreceptor-mediated HIV-1 infection and not on the interaction of the virus with DC-SIGN The evidence that HIV-1 does not effectively bind
to DC-SIGN in the presence of SE argues against a major role of this C-type lectin in sexual transmission of HIV-1
Figure 7 The HIV enhancing activity of individual SE samples
cor-relates with SEVI concentrations (A) Enhancement of HIV-1
infec-tion by SE samples from 14 different donors R5 HIV-1 stocks were
mixed with 10% (v/v) of the SE samples or PBS and used for infection
of TZM-bl indicator cells Similar results were obtained using SE
sam-ples from more than 80 additional donors Reactivity of
anti-SEVI-anti-serum from guinea pigs to (B) the indicated monomeric peptides or
IVES, a peptide containing the reverse amino acid sequence of
PAP248-286 and full-length PAP (C) SEVI fibrils or (D) pooled SE spiked
with SEVI Similar results were obtained using an antiserum from
rab-bits (E) Correlation between the magnitude of enhancement of HIV-1
infection by individual SE samples and the quantity of
SEVI/PAP248-286 detectable using the anti-SEVI antiserum.
Trang 8However, it is conceivable that a potent attachment factor
like SEVI may allow HIV-1 to bypass the requirement for
cellular attachment factors In agreement with this
possi-bility, SEVI and SE drastically enhance HIV-1 infection of
T cells both directly and in trans by epithelial cells (Figure
4) [17] Thus, amyloid aggregates in SE may help the virus
at the earliest stage of infection, when it is most
vulnera-ble to elimination [8], by promoting both virion binding
to protrusions of DCs extending to the luminal surface
and by enhancing virus infection of CD4+ target cells that
become accessible through physical breaks in the
epithe-lial barrier
We have previously shown that the small precipitate
obtained after centrifugation of SE-P contains HIV
enhancing activity and a high quantity of amyloidogenic
PAP248-286 [17] Here, we show that the potency of
indi-vidual SE samples in enhancing HIV-1 infection
corre-lates with levels of SEVI (Figure 7E) This result further
supports that SEVI contributes to the enhancing effect of
SE on HIV-1 infection Additional experiments are
required to fully elucidate the underlying mechanism
However, all data are consistent with the possibility that
amyloid aggregates bind to both the virions and the cells,
thereby allowing them to overcome the repulsion
between their negatively charged membrane surfaces
[20] Relatively small amyloid aggregates like oligomers or
filaments with sizes in the lower nanometer range may
account for the bulk of the enhancing activity because
large fibrils (> 0.1 micrometer) are not detectable in SE
[17] Indeed, it has been suggested that small fibrils are
particularly active in enhancing HIV-1 infection [39]
We found that every SE sample that liquefied and thus
could be analyzed enhanced HIV-1 infection However,
our results also showed that the potency of SE in
enhanc-ing the infectiousness of HIV-1 virions varies between
different donors It has been previously shown that some
HIV-1-infected individuals transmit the virus more
effi-ciently than others [40,41] Based on the evidence that a
small subset of infected people may be responsible for a
disproportionately high number of HIV-1 transmission, it
has been suggested that "super-spreaders" may play a
sig-nificant role in the spread of the AIDS pandemic [42]
Our findings suggest that in addition to the viral load
present in the genital fluid, the differential potency of SE
to enhance the infectiousness of HIV-1 virions may play a
relevant role in the rate of virus transmission occurring
after unprotected sexual intercourse
Although our data are highly suggestive, the
impor-tance of SE and SEVI for the spread of HIV-1 in vivo
remains elusive Previous studies in the SIV/macaque
model yielded somewhat contradictory data Neildez and
colleagues observed that treating a low dose of
SIVmac251 with SE-P increased vaginal transmission
rates [43], whereas others failed to detect significant
effects of SE or SE-P on SIV transmission [44] However, the latter may be due to the fact that the animal numbers were low and the experimental conditions did not reflect
the in vivo situation, e.g., (i) the virus doses used were
several orders of magnitude higher than those usually transferred during sexual intercourse; (ii) frozen virus stocks that may contain a relatively high proportion of defective particles were used for infection and (iii) frozen SE-P was administered into the vagina prior to the inocu-lation with SIV We found that SE and SEVI efficiently
promote SIV infection in vitro and we are planning to
perform low-dose vaginal challenge studies in the SIV/ macaque model [45] to assess the effect of SE on virus
transmission in vivo.
Although SE may strongly enhance the infectiousness
of HIV, the rate of sexual virus transmission per unpro-tected vaginal intercourse is low It is conceivable that even an effective attachment factor will not allow the virus to penetrate an intact mucosal surface Further experimentation is necessary, but it is tempting to specu-late that amyloid aggregates in SE increase the stickiness
of HIV-1 virions, thereby increasing the chances of the virus attaching to and infecting target cells that may become accessible through microlesions or local inflam-mation SE itself may play a role in the recruitment of tar-get cells to the site of primary virus exposure by stimulating inflammatory cytokines that may induce transient infiltration of the cervical mucosa by leucocytes and the migration of Langerhans cells [2,13,46]
The fact that SE enhances the infectiousness of HIV-1 may have important implications for the development of preventive vaccines and microbicides It is still not fully understood whether HIV-1 transmission by sexual inter-course usually results from cell-free or cell-associated virus However, a recent study analyzing the origin of HIV-1 strains among men who have sex with men pro-vided phylogenetic evidence that seminal plasma virus is the source [47] Thus, it is conceivable that blocking SE's ability to enhance HIV-1 infection may reduce virus transmission and the first inhibitors of SEVI have already been identified [24,25,48] SE-mediated enhancement of HIV-1 infection may also affect the antiviral potency of antibodies, microbicides and antiretroviral agents In fact, recent studies suggest that seminal plasma reduces the sensitivity of HIV-1 to inhibition by microbicides [49,50], which may explain why clinical trials have thus far generally failed [51]
Conclusions
SE efficiently enhances HIV-1 infection independently of the virus strain and producer or target cell type The mag-nitude of enhancement is donor-dependent and corre-lates with the levels of SEVI The enhancing effect of SE
on HIV-1 infection should be considered in the
Trang 9develop-ment of preventive agents and the inhibition of this host
enhancing activity may help to reduce the rates of sexual
transmission of HIV/AIDS
Methods
SE and SE-F
Semen samples were collected from healthy individuals
with informed consent Individual SE samples were
obtained from the "Kinderwunschzentrum" (Goettingen,
Germany) or the UCSF Fertility Clinic (San Francisco,
USA) Pooled SE was generated from SE samples derived
from > 20 individual donors All ejaculates were allowed
to liquefy for 30 min and SE samples were stored in 1 ml
aliquots at -20°C SE-F represents the cell free
superna-tant of SE pelleted for 5 min at 10000 rpm In all
experi-ments, aliquots were rapidly defrosted, analyzed
immediately and the remainder discarded
HIV-1 variants and virus stocks
Virus stocks of NL4-3-derived V3 recombinants [29],
HIV-1 NL4-3_R5_G-Luc (encoding the secretable
Gaussia princeps luciferase in place of nef), HIV-1 virions
pseudotyped with the envelope glycoproteins from
differ-ent transmitted/founder viruses [34], HIV-2 ROD10
(kindly provided by K Strebel), HIV-2 7312 (kindly
pro-vided by G Shaw and B.H Hahn), HIV-1 SG3.1, BRU/
LAI and YU2 (obtained through the NIH AIDS Research
and Reference Reagent Program) and SIVmac239 were
generated by transient transfection of 293T cells as
described [17] After overnight incubation, the
transfec-tion mixture was replaced by 2 ml DMEM medium
sup-plemented with 10% FCS, and the cells were cultured for
36 hours Subsequently, the culture supernatant was
cen-trifuged for 5 min at 3,000 rpm to remove cell debris The
resulting virus stock was analyzed by p24 antigen ELISA
Virus stocks were either used immediately or stored in
aliquots at -80°C Testis-derived virus was obtained at day
14 post-infection of testis explants infected with HIV-1
SF162 and IIIB as described [29]
Cell culture
TZM-bl cells (obtained through the NIH ARRRP from
Dr John C Kappes, Dr Xiaoyun Wu and Tranzyme, Inc.)
and CEMxM7 cells (kindly provided by N Landau) were
cultured as previously described [17] Human PBMC
were obtained by ficoll density centrifugation and
CD14-CD4+ cells were isolated by magnetic bead separation
(Miltenyi) 5 × 105 cells were seeded in RPMI medium
(10% FCS, Pen/Strep, Glu,) in 96-well round-bottom
plates and pre-treated with SE for one hour
Effect of SE on HIV-1 infection of TZM-bl cells
Standard testing of the effect of SE on HIV infection was
performed using adherent TZM-bl reporter cells
Typi-cally, 1 × 104 cells were seeded in microtiter plates in a
volume of 280 μl medium (DMEM supplemented with 10% FCS, 100 units/mL penicillin, 100 μg/mL streptomy-cin and 50 μg/mL gentamistreptomy-cin) The following day, frozen
SE samples were thawed, briefly vortexed, diluted in Dul-becco's PBS (1×) or medium, and mixed 1/1 (v/v) with virus stocks In most experiments, SE was serially diluted 5-fold resulting in 100%, 20%, 4% and 0.8% solutions Subsequently, 40 μl of these dilutions were transferred to U-bottom microtiter plates and mixed with 40 μl of undi-luted, 10-fold and 100-fold diluted virus stocks (corre-sponding to 2.0, 0.2 and 0.02 ng p24 antigen); referred to
as "virion treatment" Dilutions of the virus stock were used to avoid over-infection of the target cells in the pres-ence of SE The concentrations of SE during virion treat-ment were thus 50%, 10%, 2%, 0.4% and 0% After 1 to 5 min incubation at RT, the HIV/SE mixtures were resus-pended twice, and 20 μl thereof were used to infect 280 μl TZM-bl cells (70% confluent) resulting in a 15-fold dilu-tion of SE and thus final SE concentradilu-tions in the cell cul-ture of 3.3%, 0.66%, 0.13%, 0.03% and 0% Final SE concentrations > 0.1% were cytotoxic when left on the cells for longer time periods To avoid this, the HIV/SE inoculum was removed after 2 hrs of incubation at 37°C and the cells were further cultivated in 200 μl fresh DMEM (supplemented with 10% FCS and antibiotics) Even under these conditions, concentrations of ≥3.3% SE reduced the viability of the cells Infection was monitored daily by light microscopy and infection rates were deter-mined upon occurrence of virus induced cytopathic effects, or after a maximum of 3 days Infection rates of TZM-bl were determined using the one-step Tropix Gal-Screen Kit (Applied Biosystems) as recommended by the manufacturer β-galactosidase activities were determined using the Orion microplate luminometer (Berthold) All values represent reporter gene activities (relative light units per second; RLU/s) derived from triplicate infec-tions minus background activities derived from unin-fected cells To analyze the effect of SE on HIV infection under conditions approximating the in vivo situation, 20
μl of virus stocks were mixed with 180 μl SE (correspond-ing to 90% SE dur(correspond-ing virion incubation) Subsequently, 2-fold serial dilutions in PBS or medium were generated and 20 μl aliquots thereof were used to infect 280 μl TZM-bl cell cultures The HIV/SE inoculum was removed 2 hrs later and infection rates were determined
as described above
Effect of SE on HIV infection of PBMCs, macrophages and CEMx-M7 cells
PBMC were isolated and cultivated as described [17] Virus stocks were treated with 10% SE or PBS, and 20 μl
of the HIV/SE mixtures were added to 280 μl PBMCs (total of 2 × 105 cells), resulting in a final SE concentration
of 0.66% After 3 hrs, the cells were transferred to
Trang 10V-shaped microtiter plates and pelleted at 1,000 rpm for 5
min Supernatants were removed and cells resuspended
in 200 μl fresh medium At 3 days post-infection, 100 μl of
the PBMC supernatants were used to infect 100 μl
TZM-bl cell cultures (1 × 104 cells) Two days later, infection
rates were determined as described above To monitor
the effect of SE on HIV infection by fluorescence
micros-copy, a total of 2 × 105 CEMx174 M7 cells containing the
GFP reporter gene under the control of the HIV-1
pro-moter were seeded in 280 μl medium and infected with
20 μl of 10% SE-treated HIV-1 After 2 hrs cells were
pel-leted and resuspended in fresh medium Aliquots of the
cultures were analyzed by UV microscopy 3 days later
Macrophages were generated by treating buffy
coat-derived monocytes for 3 days with GMCSF (10 ng/ml)
followed by MCSF (2 ng/ml) for another 3 days Cells
were then seeded at a density of 20,000 cells/well in 280 μl
RPMI containing MCSF and infected with SE-treated or
mock-treated HIV-1 NL4-3_R5_G-Luc Two hrs later, the
cells were washed to remove Gaussia luciferase
intro-duced by the virus stock and 250 μl fresh medium was
added At 3 days post-infection, 40 μl of the culture
supernatant and 40 μl of the washing control were
ana-lyzed for Gaussia luciferase activity using the
Gaussia-Juice Kit (P.J.K) as recommended by the manufacturer
Luciferase activities were determined using the Orion
microplate luminometer (Berthold) Reporter activities in
all controls were <1,000 RLU/s
Effect of SE on HIV transmission from CaSki cells to T cells
10,000 CaSki cells (ATCC #CRL-1550), an epithelial
cer-vical carcinoma cell line, were seeded in 280 μl medium
and infected the next day with 20 μl of SE- or
mock-treated HIV After 3 hrs, the inoculum was removed and
CaSki cells were co-cultivated with 5 × 104 CEMx-M7
cells in 200 μl medium Three days later, luciferase
activi-ties in cellular lysates were determined using the
luciferase assay system (Promega)
Cell Viability
The effect of SE on metabolic activity of cells was
ana-lyzed using the MTT assay under experimental
condi-tions corresponding to those used in the respective
infection assays After 3 days of incubation, 20 μl of a 5
mg/ml MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl
tetrazolium bromide, Sigma #M2003) solution was added
to cells After 3 hrs, the cell-free supernatant was
dis-carded, formazan crystals were dissolved in 100 μl
DMSO-ethanol (1:1) and OD was detected at 490/650
nM In some experiments cytotoxic effects of SE were
further analyzed using the CellTiter-Glo Luminescent
Cell Viability Assay (Promega, #G7571) as recommended
by the manufacturer
Effect of SE on DC-SIGN-mediated trans-HIV infection
Seminal plasma (SE-P) was prepared by centrifugation of pooled SE for 30 min at 1,000 g The supernatant was passed through 0.22-μm-pore-size filters 20 μl SE or
SE-P and 5-fold dilutions thereof were added to 160 μl THSE-P-
THP-1 or THP-DC-SIGN cells (2 × THP-105) or medium only After
30 min incubation at 37°C, 20 μl R5 HIV-1 (undiluted, 10-, 100- and 110-,000-fold diluted) was added and incubated for another 30 min Thereafter, cells were transferred into 96-well V-shape plates, pelleted by centrifugation, washed two times in PBS to remove unbound virus and resuspended in 100 μl medium Subsequently, 100 μl CEMx-M7 cells (1 × 105) were added After 3 days of co-cultivation, 150 μl of the cultures were pelleted, and luciferase activities in the cell lysates were detected using Promega's luciferase assay kit as recommended by the manufacturer
HIV-1 infection of cells pretreated with SE or SE-P
The experiment was performed essentially as described [15] except that virus stocks containing normalized quan-tities (0.5 ng of p24 antigen) of an R5 tropic HIV-1 NL4-3 variant were used and that infection rates and metabolic activities were measured not only after 1 but also after 3 days post-infection SE-P was generated by pelleting SE for 30 min at 1,500 g [15] TZM-bl cells (6 × 104) were seeded the day before infection The next day 50 μl of serial dilutions of SE and SE-P were added and cells were infected with 50 μl virus stocks containing 0.5 ng p24 One and three days later, infection rates and cellular ATP levels as indicator of metabolic activity were determined using the Promega luciferase assay kit or CellTiter-Glo Luminescent Cell Viability Assay We also examined the ATP levels of SE and SE-P themselves in the absence of cells under the same experimental conditions
SEVI ELISA
96 well EIA/RIA plates (Corning Incorporated) were coated with 100 μl dilutions of antigen or 100-fold dilu-tions of cell free SE-F in PBS over night at 4°C The next day, plates were rinsed twice in wash buffer (imidazole-buffered Saline with Tween 20; KPL), blocked for 2 hrs with 100 μl 1 × Roti®-Block solution (Roth) and rinsed again 5 times Thereafter, 100 μl of 50-fold dilutions of pre-immune sera or antisera derived from SEVI amyloid immunized New Zealand White female rabbits (d28; Pocono Rabbit Farms) or guinea pigs (S3; IPF-Pharma-ceuticals) were added, and then plates were incubated for
1 hr and rinsed 5 times Finally, 100 μl of 10,000 fold dilu-tions of HRP-coupled rabbit (PerkinElmer) or anti-guinea-pig antibodies (Abcam) were added, samples were incubated for 30 min, washed 5 times, and incubated with
100 μl HRP substrate (SureBlue™, KPL) Reactions were